EP2662085A1 - Regelnde Makrophagen und ihre Verwendungen - Google Patents

Regelnde Makrophagen und ihre Verwendungen Download PDF

Info

Publication number
EP2662085A1
EP2662085A1 EP12167693.6A EP12167693A EP2662085A1 EP 2662085 A1 EP2662085 A1 EP 2662085A1 EP 12167693 A EP12167693 A EP 12167693A EP 2662085 A1 EP2662085 A1 EP 2662085A1
Authority
EP
European Patent Office
Prior art keywords
cell
cells
macrophages
macrophage
patient
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP12167693.6A
Other languages
English (en)
French (fr)
Inventor
designation of the inventor has not yet been filed The
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Humboldt Universitaet zu Berlin
Original Assignee
Humboldt Universitaet zu Berlin
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Humboldt Universitaet zu Berlin filed Critical Humboldt Universitaet zu Berlin
Priority to EP12167693.6A priority Critical patent/EP2662085A1/de
Publication of EP2662085A1 publication Critical patent/EP2662085A1/de
Ceased legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4614—Monocytes; Macrophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46433—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/73—Hydrolases (EC 3.)
    • C12N2501/734—Proteases (EC 3.4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to regulatory macrophages and their uses.
  • Atopic and autoimmune diseases are characterized by undesirable inflammation that contributes to less well-being and life expectancy of human beings.
  • Atopic diseases encompass a wide range of immunologic disorders, e. g. hay fever, atopic dermatitis, allergic sinusitis, food allergies and asthma.
  • immunologic disorders e. g. hay fever, atopic dermatitis, allergic sinusitis, food allergies and asthma.
  • allergic asthma is a frequently occurring phenomenon in westernized countries. It affects 200 to 300 million people worldwide (Fallon and Mangan, 2007) and is triggered by innocuous antigens that are usually well tolerated.
  • glucocorticoides and antihistamines are most commonly used to treat allergy-related disorders.
  • Helminth parasites are eukaryotic organisms which live as extracellular parasites in other organisms to obtain nourishment thereby inflicting morbidity. In recent years, epidemiological and laboratory studies, however, showed that helminth parasites modulate the host immune system to ensure their survival and thereby exert spillover effects on unrelated immune dysfunctions. Furthermore, successful treatment of inflammatory bowel or Crolin's s disease patients with Trichuris suis or Necator americanus in clinical trials demonstrated that helminth worms can be used for therapeutic purposes (Summers et al. 2005; Croese et al., 2006). However, although the concept of helminthic therapy has been described as safe and effective, the application of living parasites always bears the risk of unwanted side effects which might be prevented by using alternative strategies, e. g. single helminth regulatory products.
  • patent application PCT/EP2007/006888 (published as WO 2008/015014 A2 ) has presented a novel approach to treat inflammatory diseases: by treating a patient with the protein cystatin derived from nematodes, inflammatory immune reactions can be suppressed. This way, for example the induction of allergic airway hyperreactivity, or the induction of allergic gastrointestinal hyperreactivity can be inhibited.
  • cystatin is an immunogenic molecule and is recognized by the immune system of the patient. Accordingly, a patient treated with cystatin may develop immune reactions against the molecule, leading to undesired side effects. A patient sensitized to cystatin by a previous cystatin treatment may react to another exposure with an immunologic overreaction that may even take the form of an allergic shock. Alternatively, if cystatin treatment is repeated later on, the intended effects may be significantly reduced, because the patient's immune system neutralizes the cystatin molecule immediately upon entry into the patient's body.
  • PCT/EP2007/006888 requires repeated administration of cystatin to the patient, meaning an increased effort on side of the patient and the risk that the therapy may fail due to deviations from the treatment schedule.
  • a macrophage cell for use in the prevention and/or treatment of an inflammatory disease in a patient, wherein, preferably, said cell has been produced by treatment with a cystatin derived from a parasitic nematode and wherein said cell differs from murine macrophages of subpopulation M2a (by a higher expression level of the gene SPHK1 and/or the gene LIGHT,) and from murine macrophages of subpopulation M2b (by a higher expression level of the gene ARG1,) as measured by real-time PCR.
  • the above-described characteristics of said cell i.e. that said cell differs from murine macrophages of subpopulation M2a (by a higher expression level of the gene SPHK1 and/or the gene LIGHT,) and from murine macrophages of subpopulation M2b (by a higher expression level of the gene ARG1,) as measured by real-time PCR, is referred to as a "hybrid phenotype".
  • said cell is an isolated cell, preferably an isolated monocyte cell or an isolated macrophage cell.
  • such measurement is carried out at a time point that lies in the range of from 6 h to 7 days, preferably at 6 h, 12 h, 24 h, 36 h, 48 h, 3 days, 4, days, 5 days, 6 days, or 7 days after said treatment with a cystatin derived from a parasitic nematode.
  • said treatment with said cystatin derived from a parasitic nematode occurs in vivo. In one embodiment, said treatment with said cystatin derived from a parasitic nematode occurs in vitro, preferably in the presence of bystander cells, more preferably in the presence of leukocytes, which are removed after the treatment.
  • said macrophage cell is an autologous cell with respect to said patient.
  • said macrophage cell is administered to said patient, preferably by intravenous administration, more preferably by intravenous injection.
  • said cell is stored in a frozen state.
  • said cell differs from macrophages of subpopulation M2a by a lower expression level of the genes Ym1 and/or RELM- ⁇ , as measured by real-time PCR, wherein, preferably, said lower expression level of Ym1 and/or RELM- ⁇ is, independently, at least 2-fold, preferably at least 5-fold, more preferably at least 10-fold, even more preferably at least 20-fold, most preferably at least 50-fold.
  • such measurement is carried out at a time point that lies in the range of from 6 h to 7 days, preferably at 6 h, 12 h, 24 h, 36 h, 48 h, 3 days, 4, days, 5 days, 6 days, or 7 days after said treatment with a cystatin derived from a parasitic nematode.
  • said higher expression level of SPHK1, LIGHT, and/or ARG1 is, independently, at least 2-fold, preferably at least 5-fold, more preferably at least about 10-fold, more preferably at least about 20-fold, more preferably at least about 30-fold, more preferably at least about 40-fold, more preferably at least 50-fold, most preferably at least 100-fold.
  • said inflammatory disease is an atopic and/or autoimmune disease, preferably selected from the group comprising allergic diseases of the respiratory organs, such as asthma, hay fever, allergic sinusitis, allergic rhinitis, more preferably asthma or hay fever, allergic diseases of the gastrointestinal system, such as food allergies, allergic diseases of the skin, such as atopic dermatitis, systemic allergic diseases, such as anaphylactic reactions, allergic encephalomyelitis, autoimmune diseases of the joints and/or skin and/or internal organs, such as rheumatoid arthritis, psoriasis, lupus erythematosus, multiple sclerosis, and inflammatory bowel diseases, such as colitis ulcerosa and Crohn's Disease.
  • allergic diseases of the respiratory organs such as asthma, hay fever, allergic sinusitis, allergic rhinitis, more preferably asthma or hay fever
  • allergic diseases of the gastrointestinal system such as food allergies, allergic diseases of the skin, such as atopic dermatitis
  • said inflammatory bowel disease is colitis, preferably colitis ulcerosa.
  • colitis as used herein, is meant to include both acute and chronic colitis.
  • said cell is a cell for use in the prevention of a seasonal allergy, preferably a cell for use in the prevention of hay fever.
  • said cell is part of a mixture of cells with a content of macrophages higher than 85%, preferably higher than 90%, more preferably higher than 95%, most preferably higher than 98%.
  • said cystatin has a sequence selected from the group comprising SEQ ID NO:1 ( Acanthocheilonema viteae cystatin L43053), SEQ ID NO: 2 ( Onchocerca volvulus cystatin M37105), SEQ ID NO:3 ( Brugia malayi cystatin), and sequences that are 70% identical, preferably 80% identical, more preferably 90% identical and, most preferably, 99% identical to any of the foregoing.
  • said cystatin is recombinant cystatin and has been produced by a prokaryotic or eukaryotic expression system.
  • said disease is associated with an increased count of eosinophil blood cells and/or with an increased level of IgE, when compared with a patient not having said disease, and said medicament, upon its administration to said patient, leads to a reduction of said increased count of eosinophil blood cells and/or to a reduction of said increased level of IgE, preferably to a count of eosinophil blood cells and/or to a level of IgE of a healthy individual.
  • said increased count of eosinophil blood cells is > 4% of all white blood cells of a patient or > 360/ ⁇ l total number of eosinophil blood cells in peripheral blood of a patient, and said increased level of IgE is > 100 kU/l serum level in an adult patient.
  • said patient is a mammal, preferably a human being.
  • said patient is an animal, preferably selected from the group comprising a canine animal, preferably a dog, a feline animal, preferably a cat, and a rodent, preferably a mouse.
  • said cell is administered to said patient by irrigation.
  • said administration occurs less than three times, preferably only once, per occurrence of said disease in said patient.
  • said parasitic nematode is parasitic to humans or animals.
  • said parasitic nematode is parasitic to canine animals, preferably dogs, or is parasitic to rodents, preferably mice, or is parasitic to feline animals, preferably cats.
  • said nematode is selected from the group comprising Onchocerca volvulus, Brugia malayi, Wuchereria bancrofti, Loa loa and Acanthocheilonema viteae, Dirofilaria immitis, Dirofilaria repens, Nippostrongylus brasiliensis and Litomosoides sigmodontis.
  • said cell is administered to said patient in combination with one or more drugs selected from the group of anti-inflammatory drugs, preferably corticosteroids, non-steroidal anti-inflammatory drugs, and/or anti-histamines.
  • drugs selected from the group of anti-inflammatory drugs, preferably corticosteroids, non-steroidal anti-inflammatory drugs, and/or anti-histamines.
  • said administration of said cell to said patient occurs in combination with another therapeutic treatment of said patient that is aimed at treating the same inflammatory disease as treated by administration of said cell.
  • said cell differs from macrophages that have not been produced by treatment with a cystatin derived from a parasitic nematode by an increased expression, preferably by a factor of 10, of the cell surface protein MHC-II, and/or by an increased expression, preferably by a factor of 10, of the cell surface protein CD40, and/or by an increased expression, preferably by a factor of 10, of the cell surface protein PDL-2, as measured by antibody staining of cell surface proteins and flow cytometric analysis.
  • such measurement is carried out at a time point that lies in the range of from 6 h to 7 days, preferably at 6 h, 12 h, 24 h, 36 h, 48 h, 3 days, 4, days, 5 days, 6 days, or 7 days after said treatment with a cystatin derived from a parasitic nematode.
  • said cell induces T cells, preferably CD4+ T cells, to produce IL-10.
  • an autologous macrophage cell for use in the prevention and/or treatment of an inflammatory disease in a patient, wherein said macrophage cell is prepared by the steps:
  • said macrophage cell after said macrophage cell has been prepared, it is administered to said patient, preferably by intravenous administration, more preferably by intravenous injection.
  • said cell is stored in a frozen state.
  • said obtaining said cell derived from the body of said patient involves one of the following:
  • said cell differs from murine macrophages of subpopulation M2a by a lower expression level of the genes Ym1 and/or RELM- ⁇ , as measured by real-time PCR, wherein, preferably, said lower expression level of Ym1 and/or RELM- ⁇ is, independently, at least 2-fold, preferably at least 5-fold, more preferably at least 10-fold, even more preferably at least 20-fold, most preferably at least 50-fold.
  • such measurement is carried out at a time point that lies in the range of from 6 h to 7 days, preferably at 6 h, 12 h, 24 h, 36 h, 48 h, 3 days, 4, days, 5 days, 6 days, or 7 days after said treatment with a cystatin derived from a parasitic nematode.
  • said higher expression level of SPHK1, LIGHT, and/or ARG1 is, independently, at least 2-fold, preferably at least 5-fold, more preferably at least about 10-fold, more preferably at least about 20-fold, more preferably at least about 30-fold, more preferably at least about 40-fold, more preferably at least 50-fold, most preferably at least 100-fold.
  • said inflammatory disease is an atopic and/or autoimmune disease, preferably selected from the group comprising allergic diseases of the respiratory organs, such as asthma, hay fever, allergic sinusitis, allergic rhinitis, more preferably asthma or hay fever, allergic diseases of the gastrointestinal system, such as food allergies, allergic diseases of the skin, such as atopic dermatitis, systemic allergic diseases, such as anaphylactic reactions, allergic encephalomyelitis, autoimmune diseases of the joints and/or skin and/or internal organs, such as rheumatoid arthritis, psoriasis, lupus erythematosus, multiple sclerosis, and inflammatory bowel diseases, such as colitis ulcerosa and Crohn's Disease.
  • allergic diseases of the respiratory organs such as asthma, hay fever, allergic sinusitis, allergic rhinitis, more preferably asthma or hay fever
  • allergic diseases of the gastrointestinal system such as food allergies, allergic diseases of the skin, such as atopic dermatitis
  • said inflammatory bowel disease is colitis, preferably colitis ulcerosa.
  • colitis as used herein, is meant to include both acute and chronic colitis.
  • said cell is part of a mixture of cells with a content of macrophages higher than 85%, preferably higher than 90%, more preferably higher than 95%, most preferably higher than 98%.
  • said cystatin has a sequence selected from the group comprising SEQ ID NO:1 (Acanthocheilonema viteae cystatin L43053), SEQ ID NO: 2 ( Onchocerca volvulus cystatin M37105), SEQ ID NO:3 ( Brugia malayi cystatin), and sequences that are 70% identical, preferably 80% identical, more preferably 90% identical and, most preferably, 99% identical to any of the foregoing.
  • said cystatin is recombinant cystatin and has been produced by a prokaryotic or eukaryotic expression system.
  • said disease is associated with an increased count of eosinophil blood cells and/or with an increased level of IgE, when compared with a patient not having said disease, and said medicament, upon its administration to said patient, leads to a reduction of said increased count of eosinophil blood cells and/or to a reduction of said increased level of IgE, preferably to a count of eosinophil blood cells and/or to a level of IgE of a healthy individual.
  • said increased count of eosinophil blood cells is > 4% of all white blood cells of a patient or > 360/ ⁇ l total number of eosinophil blood cells in peripheral blood of a patient, and said increased level of IgE is > 100 kU/l serum level in an adult patient.
  • said patient is a mammal, preferably a human being.
  • said patient is an animal, preferably selected from the group comprising a canine animal, preferably a dog, a feline animal, preferably a cat, and a rodent, preferably a mouse.
  • said administration occurs less than three times, preferably only once, per occurrence of said disease in said patient.
  • said parasitic nematode is parasitic to humans or animals.
  • said parasitic nematode is parasitic to canine animals, preferably dogs, or is parasitic to rodents, preferably mice, or is parasitic to feline animals, preferably cats.
  • said nematode is selected from the group comprising Onchocerca volvulus, Brugia malayi, Wuchereria bancrofti, Loa loa and Acanthocheilonema viteae, Dirofilaria immitis, Dirofilaria repens, Nippostrongylus brasiliensis and Litomosoides sigmodontis.
  • said cell is administered to said patient in combination with one or more drugs selected from the group of anti-inflammatory drugs, preferably corticosteroids, non-steroidal anti-inflammatory drugs, and/or anti-histamines.
  • drugs selected from the group of anti-inflammatory drugs, preferably corticosteroids, non-steroidal anti-inflammatory drugs, and/or anti-histamines.
  • said administration of said cell to said patient occurs in combination with another therapeutic treatment of said patient that is aimed at treating the same inflammatory disease as treated by administration of said cell.
  • said cell differs from macrophages that have not been treated with a cystatin derived from a parasitic nematode by an increased expression, preferably by a factor of 10, of the cell surface protein MHC-II, and/or by an increased expression, preferably by a factor of 10, of the cell surface protein CD40, and/or by an increased expression, preferably by a factor of 10, of the cell surface protein PDL-2, as measured by antibody staining of cell surface proteins and flow cytometric analysis.
  • such measurement is carried out at a time point that lies in the range of from 6 h to 7 days, preferably at 6 h, 12 h, 24 h, 36 h, 48 h, 3 days, 4, days, 5 days, 6 days, or 7 days after said treatment with a cystatin derived from a parasitic nematode.
  • said cell induces T cells, preferably CD4+ T cells, to produce IL-10.
  • the objects of the present invention are also solved by a method for prevention and/or treatment of an inflammatory disease in a patient, said method comprising the steps:
  • said inflammatory disease, said patient, said obtaining macrophage cells, said activating said macrophage cells, said cystatin, said parasitic nematode, and said administering are as defined in the embodiments above.
  • the objects of the present invention are also solved by a method of using the macrophage cell as described above for treatment of an inflammatory disease in a patient.
  • said inflammatory disease, said patient and said macrophage cell are as defined in the embodiments above.
  • the objects of the present invention are also solved by the use of a macrophage cell for the manufacture of a medicament for the treatment of an inflammatory disease in a patient.
  • said inflammatory disease, said patient, and said macrophage cell are as defined in the embodiments above.
  • cysteine proteases refers to members of a super family of inhibitors of cysteine proteases.
  • a "parasitic nematode” is a nematode that exploits a host organism for meeting its own requirements. This may involve a life of the nematode within the tissue of a host for parts or the entire life-span of the nematode. In preferred embodiments, the host of such parasitic nematode is human.
  • a sequence that has x% identity to another sequence is a sequence in which x% residues on their respective positions are identical when optimally aligned.
  • a cystatin derived from a nematode is meant to refer to any cystatin that has the sequence of a cystatin occurring in a nematode.
  • the term is not limited to a specific production method and may include isolation of the cystatin from the nematode or production of the cystatin by other methods, such as recombinant techniques or chemical synthesis.
  • the term "a cystatin derived from a nematode” is also meant to include proteins that have retained a cystatin function despite their amino acid sequence having been mutated in one or several positions by substitutions, insertions or deletions.
  • said cell is administered to said patient in combination with another drug
  • the other drug and said cell may be in the same dosage unit, or they may be administered in separate dosage units. They may be administered by the same route or different routes.
  • the expression that the "administration of said cell to said patient occurs in combination with another therapeutic treatment” is meant to include any situation wherein said cell is administered to said patient previous to, simultaneously with, subsequent to, or overlapping with said other therapeutic treatment.
  • autologous cell refers to a situation in which said cell is derived from the same patient to whom the cell is administered later on.
  • macrophage cell and “macrophage” are used interchangeably and refer to a monocyte-derived phagocyte which is not a dendritic cell or a cell that derives from tissue macrophages by local proliferation. In the body these cells are tissue specific and refer to e. g. Kupffer cells in the liver, alveolar macrophages in the lung, microglia cells in the brain, osteoclasts in the bone etc.
  • the skilled person is aware how to identify macrophage cells, how to isolate macrophage cells from the body of a human or animal, and how to characterize macrophage cells with respect to their subclass and subpopulation (Kruisbeek, 2001; Davies and Gordon 2005 a and b; Zhang et al., 2008; Mosser and Zhang, 2008; Weischenfeldt and Porse, 2008; Ray and Dittel, 2010; Martinez et al., 2008; Jenkins et al., 2011).
  • Murine macrophages can be polarized, e. g. differentiated into specific subclasses and subpopulations, such as the subclasses designated M1 and M2 macrophages (Mills et al., 2000). Whereas the term M1 is used to describe classically activated macrophages that arise due to injury or bacterial infection and IFN-y activation, M2 has been introduced as a generic term for numerous forms of macrophages activated differently than M1. The M2 classification has further been divided into subpopulations (Mantovani et al., 2004). The most representative form is M2a macrophages, which commonly occur in helminth infections by exposure to worm induced Th2 cytokines IL-4 and IL-13.
  • M2a macrophages were, among others, shown to be essentially involved in protecting the host from re-infection (Anthony et al., 2006) or in contributing to wound healing and tissue remodeling (Gordon, 2003). Another subpopulation is M2b macrophages that produce high levels of IL-10 and low levels of IL-12 but are not per se anti-inflammatory (Anderson and Mosser, 2002; Edwards et al., 2006). M2b macrophages are elicited by immune complexes that bind to Fc- ⁇ receptors in combination with TLR ligands. Finally, M2c macrophages represent a subtype elicited by IL-10, TGF- ⁇ or glucocorticoids and can be regarded as deactivated macrophages (Martinez et al., 2008).
  • Macrophages of subpopulation M2a this is meant to designate a murine macrophage cell that has been exposed to a milieu under Th2 conditions (e g. exposure to Th2 cytokines IL-4 and IL-13) and exhibits a specific phenotype by higher expression of the gene Ym1 and/or the gene CD206 and/or the gene RELM- ⁇ and/or the gene Arginase-1. Said M2a macrophages are less efficient in antigen-presentation and lack the ability to produce nitric oxide thus showing impaired microbicidal activity.
  • Th2 conditions e g. exposure to Th2 cytokines IL-4 and IL-13
  • Said M2a macrophages are less efficient in antigen-presentation and lack the ability to produce nitric oxide thus showing impaired microbicidal activity.
  • Macrophages of subpopulation M2b this is meant to designate a murine macrophage cell that has been exposed to a milieu of immune complexes in combination with TLR or TNF-alpha stimulation. Said cell is characterized through higher expression of the gene SPHK-1 and/or the gene LIGHT and/or the gene IL-10.
  • the present application refers to a macrophage cell "derived from the body of a patient". This is meant to designate that either macrophages are obtained from the body of said patient, or macrophage precursor cells are obtained from the body of said patient and subsequently differentiated into macrophage cells in vitro as described in Wahl et al. 2006; Davis and Gordon 2005; Smythies et al., 2006; Zhang et al., 2008; Mosser and Zhang, 2008.
  • obtaining a cell is meant to include both obtaining of said cell in pure form and obtaining a cell mixture that contains said cell.
  • a cell has been produced by treatment with cystatin is meant to refer to a situation where either with the cell or a precursor of said cell or an organism in which said cell or a precursor of said cell resided at the time has been subjected to cystatin treatment.
  • activating a macrophage cell refers to a process in which the macrophage cell is subjected to a treatment that results in changes to the characteristics of said macrophage cell, such as changes in the expression of certain marker genes.
  • in vitro refers to a situation where something takes place outside of an organism. If used in connection with cells being subjected to a treatment, such cells may be either in culture or not, they may have been outside of an organism for any length of time, and they may remain outside of an organism after the treatment for any length of time.
  • bystander cells refers to all cell types other than macrophages.
  • isolated macrophage-precursors refers to a situation where the macrophage-precursors are being enriched compared to other types of cells.
  • the enrichment may be to a purity of higher than 85%, preferably higher than 90%, more preferably higher than 95%, most preferably higher than 98%, wherein these percentages refer to the number of macrophage-precursors compared to the total number of cells.
  • expression refers to the process by which the information encoded in a DNA sequence is first converted into an RNA sequence (transcription), and then further into a protein sequence (translation). The term is used to refer to either the transcription step or the translation step individually, or both steps together.
  • a "higher expression level" of a certain gene is meant to designate that one cell type or cellular situation differs from another cell type or cellular situation by having, due to an increased expression of that gene, an increased level of the RNA and/or the protein encoded by that gene.
  • Such an increased expression level can for example be detected by real-time PCR, if an increased level of an RNA is to be detected, or, if the encoded protein is a cell surface protein, by antibody staining of the cell surface protein and flow cytometric analysis.
  • SPHK1 "LIGHT”, “ARG1”, “Ym1”, RELM- ⁇ are gene designations for mouse genes.
  • inflammatory disease refers to diseases which are characterized by the presence of undesirable inflammation and inflammatory symptoms.
  • the term includes both atopic and autoimmune diseases.
  • inflammatory diseases in the sense of the present application are allergic diseases of the respiratory organs, such as asthma, hay fever, allergic sinusitis, allergic rhinitis, of the gastrointestinal system, such as food allergies, of the skin, such as atopic dermatitis, systemic allergic diseases, such as anaphylactic reactions, allergic encephalomyelitis, autoimmune diseases of the joints and/or skin and/or internal organs, such as rheumatoid arthritis, psoriasis, lupus erythematosus, multiple sclerosis, and inflammatory bowel diseases, such as colitis ulcerosa and Crohn's Disease.
  • the present inventors have now surprisingly found that treatment of a patient with cystatin derived from parasitic nematodes results in the formation of a special population of regulatory macrophages that have immunosuppressive function and are characterized by a specific expression pattern of certain marker genes.
  • a population of regulatory macrophages with the same characteristics is formed when macrophages are treated with cystatin derived from nematodes in vitro. Macrophages of this population are sufficient to suppress major symptoms of allergic airway disease and colitis.
  • the immunoregulatory function of the suppressive macrophage is conveyed by a soluble factor and is associated with the induction of IL-10 producing T cells.
  • Panel A shows the detection of total and allergen-specific levels of serum IgE or OVA-specific IgG1 and IgG2a in recipient mice.
  • Panel B shows the local cytokine response in BAL (bronchioalveolar lavage)-fluid measured by quantification of IL-4, IL-5, IL-13 and IL-10 by ELISA (enzyme-linked immunosorbent assay).
  • Panel C shows the systemic cytokine response in culture supernatants of allergen restimulated splenocytes.
  • Panel D shows cell counts for inflammatory cells in BAL-fluid.
  • Panel E shows histological stainings of representative lung bronchiole sections with periodic acid schiff (PAS) and hematoxylin eosin (HE).
  • the samples “AvCys PEC (wildtype)” and “AvCys PEC (IL-10 -/-)” show reduced levels of PAS and HE staining around the lumen compared to the control samples "cntrl. protein PEC” and “denat. AvCys PEC”.
  • the figure illustrates reduced mucus production (PAS staining) and reduced infiltration of inflammatory cells (HE staining) into the airways of asthmatic mice (Ova model) after injection of AvCystatin primed peritoneal exudate cells (PEC) from wild type mice or IL-10 deficient mice. Transfer of AvCystatin primed PEC into asthmatic mice is able to soften asthma symptoms. The effect is not mediated by IL-10 derived from primary primed AvCystatin PEC. Bars, 100 ⁇ m.
  • Figure 2 shows that macrophages ameliorate allergic inflammation in mice.
  • Macrophages were purified from PEC of AvCystatin or control pre-treated mice 18 hours post injection and transferred in OVA-sensitized mice.
  • Panel A shows the levels of total and allergen-specific IgE, OVA-IgG1 and OVA-IgG2a in recipient mice.
  • Panel B shows the concentrations of IL-4, IL-5, IL-13 and IL-10 in BAL-fluid.
  • Panel C shows Th2 cytokine levels in culture supernatants of allergen restimulated splenocytes.
  • Panel D shows the result of an analysis of BAL-fluid for total cells, eosinophils, monocytes/macrophages and lymphocytes.
  • Panel E shows the result of a histological analysis of mucus production and inflammatory cell influx by PAS and HE staining.
  • PAS staining reduced levels of PAS and HE staining are observed around the lumen compared to the sample "denat. AvCys-M ⁇ ”.
  • the figure illustrates a reduced mucus production (PAS staining) and reduced infiltration of inflammatory cells (HE staining) into the airways of asthmatic mice (Ova model) after injection of AvCystatin macrophages from wild type mice. Transfer of AvCystatin primed macrophages into asthmatic mice is able to soften asthma symptoms. Bars, 100 ⁇ m.
  • Panel F shows detection of CFSE+ (carboxyfluorescein succinimidyl ester positive) macrophages in spleen and PBLN (peribronchial lymph nodes) by flow cytometry analysis on day 31.
  • Figure 3 shows a phenotypic characterisation of AvCystatin elicited murine macrophages.
  • Macrophages were treated in vitro with AvCystatin or denatured AvCystatin and analysed for gene expression.
  • mice were intraperitoneally injected with 20 ⁇ g AvCystatin or control treatment, macrophages purified from isolated PEC and determined for gene expression.
  • Panel A shows the result of real-time PCR analyses for expression of selected murine macrophage marker genes in vitro. Results (mean ⁇ SEM) are representative of three independent experiments.
  • Panel B shows the result of transcriptional profiling of macrophages isolated from AvCystatin or control pre-treated mice. Results (means ⁇ SEM) are representative of two independent experiments. Normalized data are expressed as fold induction compared to untreated control mice. For each group, macrophages from 8-10 animals were pooled and gene expression analysed in triplicate values.
  • Panel C shows the result of a surface marker analysis for expression of MHC-II, co-stimulatory molecules CD40, CD80, CD86 and Fc ⁇ III/Fe ⁇ II (CD16/32), PDL-1, PDL-2 and ICAM- 1 in F480+/CD11b+ peritoneal macrophages after i.p. (intraperitoneal) treatment of mice with AvCystatin or controls for 18h. Data from one representative of three independent experiments. Cells were pooled from 5-6 mice per group.
  • Figure 4 shows that AvCystatin derived regulatory macrophages transmit protective effects in DSS-induced colitis.
  • AvCystatin or control pre-treated macrophages were intravenously transferred in mice one day after access to DSS.
  • Panel A shows the result of a histological analysis of distal colon sections by HE staining and scoring.
  • the figure shows HE staining of distal colon sections demonstrating less epithelial and mucosal damage as well as reduced influx of inflammatory cells after transfer of AvCystatin-treated macrophages compared to transfer of control-macrophages that shows no protective effects or DSS treatment without cell transfer.
  • Panel B shows the result of an analysis of changes in body weight.
  • Panel C shows the result of an analysis of changes in colon length.
  • Figure 5 shows that AvCystatin-macrophages in vivo and in vitro induce IL-10-producing CD4+ T cells.
  • Panel A CD4+ T cells were purified from the spleen of healthy mice or allergic animals after treatment with AvCystatin- or control-macrophages. IL-10 RNA (upper panel) and IL-10 protein (lower panel) was measured after restimulation with either ovalbumin or ConA (Concanavalin A) Results (mean ⁇ SEM) are shown from one of two independent experiments with 3-6 mice per group. Normalized data are expressed as fold induction compared to healthy control mice. **, P ⁇ 0.01.
  • Panel B Detection of IL-10 in culture supernatants after co-culture of in vivo generated AvCystatin or control-macrophages with ovalbumin-transgenic CD4+ T cells and ovalbumin peptide pre-treated DC (dendritic cells). Assays were performed in a mixed population and transwell approach or CD4+ T cells and DCs were alternatively incubated with macrophage supernatants (M ⁇ -S).
  • Panel C shows intracellular staining of IL-10 in CD4+ T cells after in vitro co-culture with DC and macrophages. Data is presented as mean ⁇ SEM for one representative of at least three independent experiments. Triplicate analysis was performed for pooled cells from 3-6 mice.*,P ⁇ 0.05;**,P ⁇ 0.01;***,P ⁇ 0.001.
  • Figure 6 shows schemes of the disease models of ovalbumin-induced airway inflammation and DSS (dextran sulfate sodium)-induced colitis.
  • Panel A shows a scheme of the ovalbumin-induced airway inflammation disease model.
  • Animals were intraperitoneally sensitized with 20 ⁇ g/animal OVA/Alum in PBS (phosphate buffered saline) on days 0 and 14 and intranasally challenged with 50 ⁇ g/animal ovalbumin on days 28 and 29.
  • PBS phosphate buffered saline
  • Panel B shows a scheme of the DSS-induced colitis disease model. Animals were given 3% DSS in solution from day 0 to 8.
  • FIG. 7 shows that transfer of B cells from AvCystatin treated animals does not protect against ovalbumin-induced airway inflammation.
  • Purified B cells from AvCystatin or control treated mice were transferred in OVA-sensitized mice 18 h post initial treatment.
  • Panel A shows total and allergen-specific IgE, OVA-IgG1 and OVA-IgG2a.
  • Panel B shows the Th2 cytokine response in BAL-fluid.
  • Panel C shows systemic cytokine levels in splenocyte culture supernatants after restimulation with ovalbumin.
  • Panel D shows inflammatory cells in BAL-fluid.
  • Panel E shows PAS and HE staining of lung sections.
  • the levels of PAS and HE staining observed around the lumen are very similar between the samples "denat. AvCys B cells" and "AvCys B cells”.
  • the figure illustrates that transfer of B cells, isolated from AvCystatin primed pertoneal exudate cells, has no effect on mucus production (PAS staining) and infiltration of inflammatory cells (HE staining) into the airways of asthmatic mice (Ova model).
  • AvCystatin primed B cells do not contribute to the amelioration of asthma symptoms in a mouse model of asthma. Bars, 100 ⁇ m.
  • Figure 8 shows additional gene expression data on macrophage phenotyping (transcriptional profiling of further gene markers of AvCystatin elicited macrophages).
  • Panel A shows the result of real-time PCR analyses for expression of iNOS, IL-6, CCL1, TGF- ⁇ , Ym1 and RELM- ⁇ in vitro.
  • Panel B shows the result of real-time PCR analyses for expression of iNOS, IL-6, CCL1, TGF- ⁇ , Ym1 and RELM- ⁇ ex vivo.
  • Results are representative of three (Panel A) and two (Panel B) independent experiments. Results are shown as means ⁇ SEM. Normalized data are expressed as fold induction compared to untreated control mice. For each group, macrophages from 8-10 animals were pooled and gene expression analysed in triplicate values.
  • SEQ ID NO:1 is the protein sequence of cystatin of Acanthocheilonema viteae (cystatin L43053)
  • SEQ ID NO:2 is the protein sequence of cystatin of Onchocerca volvulus (cystatin M37105)
  • SEQ ID NO:3 is the protein sequence of cystatin of Brugia malayi.
  • the PCR fragments were cloned into a T-overhang vector (pGEM-T Easy Vector Systems; Promega, Madison, WI) and further subcloned into the Eco-RI site of an expression vector yielding polypeptides with a leader of 6 histidines (pET-28 System; Novagen, Madison, USA).
  • the plasmids were transformed into competent E. coli BL21 cells.
  • AvCystatin and control protein were expressed in E. coli and purified by affinity chromatography as described above. Endotoxin levels for purified proteins were below 0.1 endotoxin units/ml (EU/ml).
  • EU/ml endotoxin units/ml
  • mice were sensitized upon intraperitoneal injections on days 0 and 14 with 20 ⁇ g ovalbumin (Sigma-Aldrich) emulsified in 2 mg aluminum hydroxide (Inject Alum, Pierce) in 0.2 ml PBS. Airway challenge was performed with intranasal application of 50 ⁇ g ovalbumin per animal on days 28 and 29. Na ⁇ ve control mice were sensitized with aluminum hydroxide in PBS and challenged with PBS instead of ovalbumin. On day 31 animals were sacrificed. Blood samples were collected for serum antibody analysis and the trachea flushed with a protease inhibitor cocktail (Roche completeTM Mini protease inhibitor tablets dissolved in PBS) for cytokine detection.
  • a protease inhibitor cocktail Roche completeTM Mini protease inhibitor tablets dissolved in PBS
  • Cytospin slides were prepared from 100 ⁇ l BAL fluid, stained with the Hemacolor® staining set for microscopy (Merck) and analysed in a blinded fashion to determine cell numbers. Moreover, lungs were fixed in 3.5% Formalin for histological analysis of mucus production in bronchioles (PAS staining) and inflammatory cell infiltration in lung tissue (HE staining). Stained slices were analyzed via light microscopy (Leica DFC480 camera and Leica Application Suite Software version 2.8.1).
  • Splenocytes from recipient mice were restimulated four days in 96 well plates with 50 ⁇ g/ml OVA or 2.5 ⁇ g/ml ConA in RPMI supplemented with 10% FCS (fetal calf serum), 1 mM LGlutamin, 100 U/ml Penicillin G and 100 ⁇ g/ml Streptomycin (cRPMI). Supernatants were frozen at - 20 °C for cytokine measurement. Cytokine levels were assessed for IL-4, IL-5, IL-13 and IL-10 by sandwich ELISA according to the manufacturer's protocol (eBioscience).
  • ELISA plates were coated with 10 ⁇ g/ml ovalbumin at 4°C overnight and unspecific binding blocked by 3% BSA (bovine serum albumin) in PBS for 1 h. After incubation with mouse serum, HRP(horseradish peroxidase)-IgE (rat antimouse IgE heavy chain HRP (MCA419P), Serotec) was used as detection antibody and specific binding visualized by application of the HRP-substrate TMB (Tetramethylbenzidine).
  • BSA bovine serum albumin
  • HRP(horseradish peroxidase)-IgE rat antimouse IgE heavy chain HRP (MCA419P), Serotec
  • OVA-specific IgG1 and IgG2 were measured in an analogical manner whereas alkaline phosphatase conjucated antibodies (anti-mouse IgG1 (Gamma 1 chain) and anti-mouse IgG2a (Gamma 2a chain), both Rockland Immunochemicals) were used as detection antibodies and nitrophenylphosphat (Sigma-Aldrich) as phosphatase substrate.
  • IgE- purchasable IgE- (purified mouse IgE, ⁇ isotype control, BD Biosciences), IgGl-(purified mouse IgG1, ⁇ isotype control, BD Biosciences) and IgG2a- (purified mouse IgG2a, ⁇ isotype control, BD Biosciences) standards were used.
  • IgGl- purified mouse IgG1, ⁇ isotype control, BD Biosciences
  • IgG2a- purified mouse IgG2a, ⁇ isotype control, BD Biosciences
  • mice were intraperitoneally treated with 20 ⁇ g recombinant AvCystatin or control treatment. 18 hours later, peritoneal exudate cells (PEC) were isolated and either directly used for cell transfer (2x10 6 ) or further purified for macrophages and B cells. Purification of B cells was performed by incubating PEC with anti-CD19 MicroBeads (Miltenyi Biotec) and cell separation upon autoMACSTM.
  • PEC peritoneal exudate cells
  • Macrophages were purified by incubating PEC with biotinylated antibodies anti-CD3 ⁇ (clone 145-2C11, Miltenyi Biotec), anti-CD19 (clone eBiolD3, eBioscience), anti-CD11c (clone N418, eBioscience), anti-Gr1 (clone RB6-8C5, kind gift of the DRFZ) and anti-Dx5 (clone Dx5, eBioscience). Addition of 40 ⁇ g/ml anti-mouse Fc ⁇ R (clone 2.4G2, DRFZ) was used to avoid non-specific binding.
  • PEC were seeded in plastic tissue-culture dishes (24-well plates) and incubated at 37°C and 5% CO 2 in Dulbecco's modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml Penicillin G and 100 ⁇ g/ml Streptomycin (cDMEM). After 4 hours, non adhering cells were removed by washing with fresh cDMEM. Remaining macrophages were cultured over night in cDMEM and stimulated on the following day with either 0.5 ⁇ M recombinant AvCystatin or control treatments.
  • DMEM Dulbecco's modified Eagle's Medium
  • cDMEM penicillin G
  • cDMEM Streptomycin
  • Macrophages were washed with Dulbecco ⁇ s PBS after indicated timepoints and frozen at - 80°C in lysis buffer (analytikjena).
  • lysis buffer analytikjena
  • macrophages were purified from isolated PEC of AvCystatin or control treated animals 18 hours post application as described above. Purified macrophages were lysated and frozen at -80°C. Surface marker expression was determined for CD11b+F4/80+ macrophages 18 h post i.p.
  • anti-MHC-II-FITC (clone M5/114.15.2, eBioscience), anti-CD274-PE (clone MIH5, BD Biosciences), anti-CD273-PE (clone TY25, BD Biosciences), anti-CD40-PE (clone 1C10, eBioscience), anti-CD86-biotin (clone GL1, eBioscience), streptavidin-PE-Cy7 (clone Streptavidin, BD Biosciences), anti-CD16/32-APC (clone 2.4G2, kind gift from the DRFZ), anti-CD80-PE (clone 16-10A1, eBioscience) and anti-ICAM-1-APC (clone KAT1, kind gift from the DRFZ).
  • Non-specific binding was prevented by the addition of 20 ⁇ g/ml anti-mouse Fc ⁇ R (clone 2.4G2, kind from the DRZF). After antibody binding cells were washed with Dulbecco's PBS supplemented with 0.2% BSA and 10 mM EDTA and flow cytometry analysis performed by using FACS-Canto cytometer (BD) and FlowJo Software version 8.5.3.
  • RNA isolation and reverse transcription was performed by using the innuPREP RNA kit (Analytik Jena) and the High Capacity RNA-to-cDNA Kit (Applied Biosystems) according to the manufacturer's instructions.
  • Real-time PCR was performed with the ABI Prism 7300 Sequence Detection System (Applied Biosystems) using SYBR Green PCR reagents (FastStart Universal SYBR Green Master (Rox), Roche) following the manufacturer's guidelines.
  • CD4+ were purified from spleens of ovalbumin-sensitized and -challenged mice seven days after adoptive transfer of macrophages.
  • CD4+ T cells were isolated by using the CD4+ T cell isolation kit according to the manufacturer's guidelines (Miltenyi Biotec, Bergisch Gladbach, Germany). 3x10 6 cells were stimulated in a 96 well plate (Coming Costar, Bodenheim, Germany) with ovalbumin (50 ⁇ g/ml) or ConA (2.5 ⁇ g/ml) for 96 hours in cRPMI. Supernatants were collected and frozen for IL-10 cytokine ELISA (BD Biosciences, Heidelberg, Germany). Cells were treated with lysis buffer for RNA extraction (Analytikjena, Jena, Germany), RNA isolated from cell lysates, transcribed into cDNA and IL-10 expression determined by quantitative real time-PCR.
  • the bone marrow was flushed with RPMI supplemented with 100 U/ml Penicillin G and 100 mg/ml Streptomycin from the femur and tibia of hind legs and collected in a pre-cooled sterile petri dish (Sarstedt, Nümbrecht, Germany).
  • the marrow was dissociated by slightly pipetting up and down and cells filtered through a 70 ⁇ M nylon cell strainer (BD Biosciences, Heidelberg, Germany). After centrifugation (1200 rpm, 4°C, 10 min), erythrocyte lysis was performed for 5 minutes on ice.
  • Splenocytes were prepared from the spleen of euthanized BALB/c DO11.10 TGN mice and CD4+ T cells purified by using the CD4+ T cell isolation kit according to the manufacturer's description (Miltenyi Biotec, Bergisch Gladbach, Germany). Purification of macrophages from filarial cystatin (20 ⁇ g/mouse) or control-treated BALB/c mice was performed as described earlier on.
  • CD4+ T cells were incubated with macrophages and dendritic cells in a ratio of 5:1:1.
  • the assays were performed in a mixed population and a transwell approach separating the macrophages from dendritic cells and T cells by 1.0 ⁇ m filter membranes (greiner bio-one, Frickenhausen, Germany).
  • CD4+ T cells and dendritic cells were additionally incubated in the presence of fresh macrophage supernatants. Therefore, macrophages were separately cultured for 48 hours and the complete supernatant subsequently added to CD4+ T cells and dendritic cells. Culture supernatants were finally collected and analyzed for IL-10 by ELISA (BD Biosciences, Heidelberg, Germany).
  • Remaining cells were intracellularly stained for IL-10. Therefore, cells were stimulated with 1 ⁇ g/ml PMA/Ionomyocin (Sigma-Aldrich, Steinheim, Germany) for three hours in fresh cRPMI at 37°C and 5% CO 2 . After half an hour of incubation Brefeldin A (Sigma-Aldrich, Steinheim, Germany) was added in a concentration of 1 ⁇ g/ml. Cells were washed with FACS buffer, stained with anti-CD4-PerCP-eF710 (clone RM4-5; eBioscience, Frankfurt am Main, Germany) and incubated for 15 minutes on ice.
  • PMA/Ionomyocin Sigma-Aldrich, Steinheim, Germany
  • Colitis was induced by providing 3% DSS (MP Biomedicals, Lot No. 6683K) solution to female CL57/BL6 mice for 8 days.
  • 3 x 10 6 purified macrophages from AvCystatin or control treated CL57/BL6 were administered i.v. on day 1 of access to DSS.
  • Changes in body weight were determined by weighing mice during d0 and d8. After dissection on day 8 colon length was measured and parts of distal colon fixed in 3.5% paraformaldehyde to evaluate the histological damage score upon HE staining.
  • a mouse model of ovalbumin-induced airway inflammation was used.
  • Donor cells were isolated 18h after i.p.-treatment with AvCystatin or control injections with denatured AvCystatin or an unrelated control protein and transferred four days prior to challenge with ovalbumin in recipient mice ( Fig. 6, A ).
  • Administration of AvCystatin-primed PEC led to drastically reduced OVA-specific and total IgE levels compared to control treatments.
  • OVA-specific IgG1 and IgG2a responses were not altered by transfer of AvCystatin-treated PEC ( Fig. 1, A ).
  • Regulators macrophages induced by AvCystatin protect mice from airway inflammation.
  • AvCystatin-induced macrophages from the peritoneum of donor mice 18h after treatment with AvCystatin were sorted and transferred. Purified peritoneal B cells after AvCystatin treatment were used as an additional control.
  • OVAspecific and total-IgE antibody responses were significantly suppressed in mice receiving the AvCystatin-induced regulatory macrophages compared to control treated macrophages ( Fig. 2, A ) or B cells from AvCystatin treated animals that showed no effect ( Fig. 7 ).
  • Th2-cytokines in BAL and spleen were hampered but IL- 10 levels significantly increased ( Fig. 2 , B and C). Eosinophil infiltration to the lungs was suppressed by 43% upon treatment with AvCystatin-induced regulatory macrophages ( Fig.
  • CFSE labelled cells were transferred intravenously 4 days prior to challenge with the allergen and their distribution in different tissues was analyzed by flow cytometry. CFSE labelled macrophages were recovered mainly in the spleen but were also detected to a lower amount in peribronchial lymph nodes ( Fig. 2, F ).
  • AvCystatin treatment marker genes of the four common macrophage phenotypes were analyzed: M1, M2a, M2b and M2c (Mantovani et al., 2004; Martinez et al., 2008).
  • the AvCystatin-induced macrophages were at first characterized in vitro.
  • AvCystatin treatment induced early expression of pro- and anti-inflammatory genes IL-12/23p40, TNF- ⁇ , iNOS, IL-6 and IL-10 ( Fig. 3 , A and 8, A).
  • AvCystatin treatment stimulated a specific expression profile in macrophages with an early and transient induction of pro- and anti-inflammatory genes followed by a switch to a macrophage phenotype with a specific, novel expression profile.
  • AvCystatin-elicited macrophages showed an up-regulation of MHC-II, CD40, CD80, Fc ⁇ III/Fc ⁇ II (CD16/32), PDL-1 (CD274), PDL-2 (CD273), ICAM-1 and low levels of CD86 ( Fig. 3, C ).
  • AvCystatin-induced regulatory macrophages prevent inflammation also in colitis
  • the present inventors investigated whether AvCystatin-induced macrophages had any effects on an experimental model of colitis in mice.
  • Treatment with AvCystatin-primed macrophages once led to an amelioration of colitis as measured by histological scoring of colon sections ( Fig. 4, A ), significant differences in body weight loss ( Fig. 4, B ) as well as in colon length ( Fig. 4, C ).
  • CD4+ T cells (purity ⁇ 95%) were purified from the spleens of healthy control mice or ovalbumin-sensitized and -challenged animals treated with denatured AvCystatin- or AvCystatin-macrophages prior to intranasal provocation.
  • antigen ovalbumin
  • ConA mitogen

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Cell Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Engineering & Computer Science (AREA)
  • Mycology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Hematology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Virology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Oncology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
EP12167693.6A 2012-05-11 2012-05-11 Regelnde Makrophagen und ihre Verwendungen Ceased EP2662085A1 (de)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP12167693.6A EP2662085A1 (de) 2012-05-11 2012-05-11 Regelnde Makrophagen und ihre Verwendungen

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
EP12167693.6A EP2662085A1 (de) 2012-05-11 2012-05-11 Regelnde Makrophagen und ihre Verwendungen

Publications (1)

Publication Number Publication Date
EP2662085A1 true EP2662085A1 (de) 2013-11-13

Family

ID=46062136

Family Applications (1)

Application Number Title Priority Date Filing Date
EP12167693.6A Ceased EP2662085A1 (de) 2012-05-11 2012-05-11 Regelnde Makrophagen und ihre Verwendungen

Country Status (1)

Country Link
EP (1) EP2662085A1 (de)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015084730A1 (en) * 2013-12-02 2015-06-11 The Board Of Trustees Of The Leland Stanford Junior University Regulatory macrophages as a cell-based immunomodulatory therapy in pulmonary hypertension and right ventricular dysfunction
CN105274054A (zh) * 2015-09-07 2016-01-27 中山大学中山眼科中心 体外诱导调节性巨噬细胞及其制备方法和应用
EP3372675A1 (de) * 2017-03-09 2018-09-12 Université de Liège Verfahren zur in-vitro-differenzierung von monozyten gegenüber regelnden makrophagen
EP3456819A1 (de) * 2017-09-13 2019-03-20 Trizell GmbH Regulatorische makrophagen zur behandlung von angiopathien
CN113897337A (zh) * 2021-05-31 2022-01-07 中国科学院深圳先进技术研究院 一种调节巨噬细胞极化状态的方法
WO2022252097A1 (zh) * 2021-05-31 2022-12-08 中国科学院深圳先进技术研究院 一种调节巨噬细胞极化状态的方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003044037A2 (en) * 2001-11-21 2003-05-30 Yeda Research And Development Co Ltd. Process for the manufacture of human mononuclear phagocytic leukocytes
WO2008015014A2 (en) 2006-08-04 2008-02-07 Humboldt-Universität Zu Berlin Cystatin from nematodes in the treatment of autoimmune or allergic diseases

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003044037A2 (en) * 2001-11-21 2003-05-30 Yeda Research And Development Co Ltd. Process for the manufacture of human mononuclear phagocytic leukocytes
WO2008015014A2 (en) 2006-08-04 2008-02-07 Humboldt-Universität Zu Berlin Cystatin from nematodes in the treatment of autoimmune or allergic diseases

Non-Patent Citations (35)

* Cited by examiner, † Cited by third party
Title
ANDERSON, C. A.; D.M. MOSSER: "A novel phenotype for activated macrophages: the type II activated macrophage", J. LEUKOC. BIOL., vol. 72, 2002, pages 101 - 106
ANTHONY, R.M.; J.F. URBAN, JR; F. ALEM; H.A. HAMED; C.T. ROZO; J.L. BOUCHER; N. VAN ROOIJEN; W.C. GAUSE: "Memory TH2 cells induce alternatively activated macrophages to mediate protection against nematode parasites", NAT MED., vol. 12, no. 8, 2006, pages 955 - 960
AVL7; HARTMANN, S.; KYEWSKY, B.; SONNENBURG, B.; LUCIUS, R.: "A filarial cysteine protease inhibitor downregulates T cell proliferation and enhances IL-10 production", EUR. J. IMMUNOL., vol. 27, 1997, pages 2253 - 2260
BACH JF: "The effect of infections on susceptibility to autoimmune and allergic diseases", N ENGL J MED., vol. 347, no. 12, 2002, pages 911 - 20
BREM-EXNER BEATE G ET AL: "Macrophages driven to a novel state of activation have anti-inflammatory properties in mice", JOURNAL OF IMMUNOLOGY, vol. 180, no. 1, January 2008 (2008-01-01), pages 335 - 349, XP002684131, ISSN: 0022-1767 *
CROESE J; O'NEIL J; MASSON J; COOKE S; MELROSE W; PRITCHARD D; SPEARE R: "A proof of concept study establishing Necator americanus in Crohn's patients and reservoir donors", GUT, vol. 55, no. 1, 2006, pages 136 - 37
DAVIES JQ; GORDON S: "Isolation and culture of human macrophages", METHODS MOL BIOL., vol. 290, 2005, pages 105 - 16
DAVIES JQ; GORDON S: "Isolation and culture of murine macrophages", METHODS MOL BIOL., vol. 290, 2005, pages 91 - 103
EDWARDS, J.P.; X. ZHANG; K.A. FRAUWIRTH; D.M. MOSSER: "Biochemical and functional characterization of three activated macrophage populations", J LEUKOC BIOL., vol. 80, no. 6, 2006, pages 1298 - 307
FALLON PG; MANGAN NE: "Suppression of TH2-type allergic reactions by helminth infection", NAT REV IMMUNOL., vol. 7, no. 3, 2007, pages 220 - 30
GORDON, S.: "Alternative activation of macrophages", NAT REV IMMUNOL., vol. 3, no. 1, 2003, pages 23 - 35
HARTMANN S ET AL: "Modulation of host immune responses by nematode cystatins", INTERNATIONAL JOURNAL OF PARASITOLOGY, PERGAMON PRESS, GB, vol. 33, no. 11, 1 January 2003 (2003-01-01), pages 1291 - 1302, XP002413960, ISSN: 0020-7519, DOI: 10.1016/S0020-7519(03)00163-2 *
HAWRYLOWICZ CM; O'GARRA A: "Potential role of interleukin-l0-secreting regulatory T cells in allergy and asthma", NAT REV IMMUNOL., vol. 5, no. 4, 2005, pages 271 - 283
HUTCHINSON JAMES A ET AL: "Human regulatory macrophages", METHODS IN MOLECULAR BIOLOGY, HUMANA PRESS, vol. 677, 1 January 2011 (2011-01-01), pages 181 - 192, XP009162998, ISSN: 1940-6029, DOI: 10.1007/978-1-60761-869-0_13 *
JENLDNS SJ; ALLEN JE: "Similarity and diversity in macrophage activation by nematodes, trematodes, and cestodes", J BIOMED BIOTECHNOL., 2010
JENLDNS SJ; RUCKERL D; COOK PC; JONES LH; FINKELMAN FD; VAN ROOIJEN N; MACDONALD AS; ALLEN JE: "Local macrophage proliferation, rather than recruitment from the blood, is a signature ofTH2 inflammation", SCIENCE, vol. 332, no. 6035, 2011, pages 1284 - 88
KLOTZ, C.; T. ZIEGLER; A.S. FIGUEIREDO; S. RAUSCH; M.R. HEPWORTH; N. OBSIVAC; C. SERS; R. LANG; P. HAMMERSTEIN; R. LUCIUS: "A Helminth Immunomodulator Exploits Host Signaling Events to Regulate Cytoldne Production in Macrophages", PLOS PATHOGENS, vol. 7, no. 1, 2011, pages EL001248
KRUISBEEK AM: "Curr Protoc Immunol.", 2001, article "Isolation of mouse mononuclear cells"
LIVAK KJ; SCHMITTGEN TD: "Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method", METHODS, vol. 25, no. 4, 2001, pages 402 - 08
MANTOVANI, A.; A. SICA; S. SOZZANI; P. ALLAVENA; A. VECCHI; M. LOCATI: "The chemokine system in diverse forms of macrophage activation and polarization", TRENDS IMMUNOL., vol. 25, no. 12, 2004, pages 677 - 86, XP004628806, DOI: doi:10.1016/j.it.2004.09.015
MARTINEZ, F.O.; A. SICA; A. MANTOVANI; M. LOCATI: "Macrophage activation and polarization", FRONT BIOSCI., vol. 13, 2008, pages 453 - 61
MIA M S ET AL: "Inducing regulatory macrophages through dissecting the spectrum of macrophage activation kinetically and genetically", CYTOKINE, ACADEMIC PRESS LTD, PHILADELPHIA, PA, US, vol. 48, no. 1-2, 1 October 2009 (2009-10-01), pages 134 - 135, XP026624928, ISSN: 1043-4666, [retrieved on 20090918], DOI: 10.1016/J.CYTO.2009.07.570 *
MILLS, C.D.; K. KINCAID; J.M. ALT; M.J. HEILMAN; A.M. HILL: "M-1/M-2 macrophages and the Thl/Th2 paradigm", J IMMUNOL., vol. 164, no. 12, 2000, pages 6166 - 73
MOSSER DM; ZHANG X.: "Curr Protoc Immunol.", 2008, article "Activation of murine macrophages"
OVL7; LUSTIGMAN S; BROTMAN B; HUIMA T; PRINCE AM; MCKERROW JH.: "Molecular cloning and characterization of onchocystatin, a cysteine proteinase inhibitor of Onchocerca volvulus", J BIOL CHEM., vol. 267, 1992, pages 17339 - 46
RAY A; DITTEL BN: "Isolation of mouse peritoneal cavity cells", J VIS EXP., vol. 28, no. 35, 2010, pages 1488
SCH6NEMEYER, A.; LUCIUS, R.; SONNENBURG, B.; BRATTIG, N.; SABAT, R.; SCHILLING, K.; BRADLEY, J.; HARTMANN, S.: "Modulation of human T cell responses and macrophage functions by onchocystatin, a secreted protein of the filarial nematode Onchocerca volvulus", J. IMMUNOL., vol. 167, 2001, pages 3207 - 3215, XP002413959
SCHNOELLER, C.; S. RAUSCH; S. PILLAI; A. AVAGYAN; B.M. WITTIG; C. LODDENKEMPER; A. HAMANN; E. HAMELMANN; R. LUCIUS; S. HARTMANN: "A helminth immunomodulator reduces allergic and inflammatory responses by induction of IL-10-producing macrophages", J IMMUNOL., vol. 180, no. 6, 2008, pages 4265 - 72
SENJU S; M HARUTA; K MATSUMURA; Y MATSUNAGA; S FUKUSHIMA; T IKEDA; K TAKAMATSU; A IRIE; Y NISHIMURA: "Generation of dendritic cells and macrophages from human induced pluripotent stem cells aiming at cell therapy", GENE THERAPY, vol. 18, no. 9, 2011, pages 874 - 83, XP055027827, DOI: doi:10.1038/gt.2011.22
SUMMERS RW; ELLIOTT DE; URBAN JF JR; THOMPSON RA; WEINSTOCK JV: "Trichuris suis therapy for active ulcerative colitis: a randomized controlled trial", GASTROENTEROLOGY, vol. 128, no. 4, 2005, pages 825 - 32, XP005110857, DOI: doi:10.1053/j.gastro.2005.01.005
THOMAS E. CREIGHTON: "Proteins, Structures and Molecular Properties, second edition,", W. H. FREEMAN AND CO.
WAHL LM; WAHL SM; SMYTHIES LE; SMITH PD: "Curr Protoc Immunol.", 2006, article "Isolation of human monocyte populations"
WEISCHENFELDT J; PORSE B: "Bone Marrow-Derived Macrophages (BMM): Isolation and Applications", 2008, CSH PROTOC.
YAZDANBAKHSH M; KREMSNER PG; VAN REE R: "Allergy, parasites, and the hygiene hypothesis", SCIENCE, vol. 296, no. 5567, 2002, pages 490 - 94, XP002430374, DOI: doi:10.1126/science.296.5567.490
ZHANG X; GONCALVES R; MOSSER DM: "Curr Protoc Immunol.", 2008, article "The isolation and characterization of murine macrophages"

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015084730A1 (en) * 2013-12-02 2015-06-11 The Board Of Trustees Of The Leland Stanford Junior University Regulatory macrophages as a cell-based immunomodulatory therapy in pulmonary hypertension and right ventricular dysfunction
CN105274054A (zh) * 2015-09-07 2016-01-27 中山大学中山眼科中心 体外诱导调节性巨噬细胞及其制备方法和应用
EP3372675A1 (de) * 2017-03-09 2018-09-12 Université de Liège Verfahren zur in-vitro-differenzierung von monozyten gegenüber regelnden makrophagen
EP3456819A1 (de) * 2017-09-13 2019-03-20 Trizell GmbH Regulatorische makrophagen zur behandlung von angiopathien
WO2019053091A1 (en) * 2017-09-13 2019-03-21 Trizell Gmbh REGULATORY MACROPHAGES FOR THE TREATMENT OF ANGIOPATHIES
US11648266B2 (en) 2017-09-13 2023-05-16 Trizell Gmbh Regulatory macrophages for treating angiopathies
CN113897337A (zh) * 2021-05-31 2022-01-07 中国科学院深圳先进技术研究院 一种调节巨噬细胞极化状态的方法
WO2022252097A1 (zh) * 2021-05-31 2022-12-08 中国科学院深圳先进技术研究院 一种调节巨噬细胞极化状态的方法

Similar Documents

Publication Publication Date Title
Duan et al. Innate signals from Nod2 block respiratory tolerance and program TH2-driven allergic inflammation
Wang et al. Interleukin-25 mediates transcriptional control of PD-L1 via STAT3 in multipotent human mesenchymal stromal cells (hMSCs) to suppress Th17 responses
EP2662085A1 (de) Regelnde Makrophagen und ihre Verwendungen
Merkley et al. Modulating T cell responses via autophagy: the intrinsic influence controlling the function of both antigen-presenting cells and T cells
Melgert et al. Macrophages: regulators of sex differences in asthma?
Gong et al. T follicular helper cell subsets and the associated cytokine IL-21 in the pathogenesis and therapy of asthma
Ando et al. Histamine-releasing factor enhances food allergy
Begum-Haque et al. Augmentation of regulatory B cell activity in experimental allergic encephalomyelitis by glatiramer acetate
De Araújo et al. PAS‐1, an Ascaris suum Protein, Modulates Allergic Airway Inflammation via CD8+ γδTCR+ and CD4+ CD25+ FoxP3+ T Cells
KR101469167B1 (ko) 봉독-ｐｌａ2를 포함하는 이상 조절 ｔ 세포 활성 저하 관련 질환의 치료 또는 예방용 약학적 조성물
Kita ILC2s and fungal allergy
Huang et al. Neutrophils regulate humoral autoimmunity by restricting interferon-γ production via the generation of reactive oxygen species
Lu et al. Interleukin‑27 ameliorates allergic asthma by alleviating the lung Th2 inflammatory environment
US20220339191A1 (en) Interleukin-27 producing b-cells and uses thereof
JP2022050478A (ja) Ｉｌｃ２細胞に関連する疾患を処置する方法
Lineburg The contribution of cytokine signals, autophagy and antigen presentation to complications associated with allogeneic stem cell transplantation
Ma The effect of Interleukin-10-differentiated dendritic cell therapy on B cell tolerance in a mouse model of asthma
Schinnerling et al. Jorge A. Soto, and Felipe Melo-Gonzalez
Patel The Role of Neutrophils and Their Proteases in Allergic Airway Disease
Dong The Immunological Roles of Pro-inflammatory Cytokine IL-32gamma and Regulatory Cytokine IL-35 in Allergic Inflammation and the Underlying Molecular Mechanisms
Chen The Innate and Adaptive Immune Regulation of Allergic Airway Inflammation
신태훈 Anti-inflammatory mechanisms of human umbilical cord blood-derived mesenchymal stem cells on atopic dermatitis and rheumatoid arthritis
Hsu The Role of Meningeal Lymphangiogenic Vessels as an Immune-Regulatory Niche during Neuroinflammation
Sokołowska et al. Allergy and Inflammation
Kummola The Effect of Allergic Cytokine Environment on Myeloid Cells

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN REFUSED

18R Application refused

Effective date: 20131214